Novel role of Rac-Mid1 signaling in medial cerebellar development.

Rac signaling impacts a relatively large number of downstream targets; however, few studies have established an association between Rac pathways and pathological conditions. In the present study, we generated mice with double knockout of Rac1 and Rac3 (Atoh1-Cre;Rac1flox/flox;Rac3-/- ) in cerebellar granule neurons (CGNs). We observed impaired tangential migration at E16.5, as well as numerous apoptotic CGNs at the deepest layer of the external granule layer (EGL) in the medial cerebellum of Atoh1-Cre;Rac1flox/flox;Rac3-/- mice at P8. Atoh1-Cre;Rac1flox/flox;Rac3-/- CGNs differentiated normally until expression of p27kip1 and NeuN in the deep EGL at P5. Primary CGNs and cerebellar microexplants from Atoh1-Cre;Rac1flox/flox;Rac3-/- mice exhibited impaired neuritogenesis, which was more apparent in Map2-positive dendrites. Such findings suggest that impaired tangential migration and final differentiation of CGNs have resulted in decreased cerebellum size and agenesis of the medial internal granule layer, respectively. Furthermore, Rac depleted/deleted cells exhibited decreased levels of Mid1 and impaired mTORC1 signaling. Mid1 depletion in CGNs produced mild impairments in neuritogenesis and reductions in mTORC1 signaling. Thus, a novel Rac-signaling pathway (Rac1-Mid1-mTORC1) may be involved in medial cerebellar development.

Congenital diaphragmatic hernia as a prominent feature of a SPECC1L-related syndrome.

Congenital diaphragmatic hernias (CDH) confer substantial morbidity and mortality. Genetic defects, including chromosomal anomalies, copy number variants, and sequence variants are identified in ~30% of patients with CDH. A genetic etiology is not yet found in 70% of patients, however there is a growing number of genetic syndromes and single gene disorders associated with CDH. While there have been two reported individuals with X-linked Opitz G/BBB syndrome with MID1 mutations who have CDH as an associated feature, CDH appears to be a much more prominent feature of a SPECC1L-related autosomal dominant Opitz G/BBB syndrome. Features unique to autosomal dominant Opitz G/BBB syndrome include branchial fistulae, omphalocele, and a bicornuate uterus. Here we present one new individual and five previously reported individuals with CDH found to have SPECC1L mutations. These cases provide strong evidence that SPECC1L is a bona fide CDH gene. We conclude that a SPECC1L-related Opitz G/BBB syndrome should be considered in any patient with CDH who has additional features of hypertelorism, a prominent forehead, a broad nasal bridge, anteverted nares, cleft lip/palate, branchial fistulae, omphalocele, and/or bicornuate uterus.

aura (mid1ip1l) regulates the cytoskeleton at the zebrafish egg-to-embryo transition.

Embryos from females homozygous for a recessive maternal-effect mutation in the gene aura exhibit defects including reduced cortical integrity, defective cortical granule (CG) release upon egg activation, failure to complete cytokinesis, and abnormal cell wound healing. We show that the cytokinesis defects are associated with aberrant cytoskeletal reorganization during furrow maturation, including abnormal F-actin enrichment and microtubule reorganization. Cortical F-actin prior to furrow formation fails to exhibit a normal transition into F-actin-rich arcs, and drug inhibition is consistent with aura function promoting F-actin polymerization and/or stabilization. In mutants, components of exocytic and endocytic vesicles, such as Vamp2, Clathrin and Dynamin, are sequestered in unreleased CGs, indicating a need for CG recycling in the normal redistribution of these factors. However, the exocytic targeting factor Rab11 is recruited to the furrow plane normally at the tip of bundling microtubules, suggesting an alternative anchoring mechanism independent of membrane recycling. A positional cloning approach indicates that the mutation in aura is associated with a truncation of Mid1 interacting protein 1 like (Mid1ip1l), previously identified as an interactor of the X-linked Opitz G/BBB syndrome gene product Mid1. A Cas9/CRISPR-induced mutant allele in mid1ip1l fails to complement the originally isolated aura maternal-effect mutation, confirming gene assignment. Mid1ip1l protein localizes to cortical F-actin aggregates, consistent with a direct role in cytoskeletal regulation. Our studies indicate that maternally provided aura (mid1ip1l) acts during the reorganization of the cytoskeleton at the egg-to-embryo transition and highlight the importance of cytoskeletal dynamics and membrane recycling during this developmental period.

The E3 ubiquitin ligase MID1/TRIM18 promotes atypical ubiquitination of the BRCA2-associated factor 35, BRAF35.

MID1/TRIM18 is a member of the TRIM family of ubiquitin E3 ligases characterized by the presence of a conserved RING-containing N-terminal tripartite motif. Mutations in the MID1 gene have been associated with the X-linked form of Opitz Syndrome, a developmental disorder characterized by midline defects and intellectual disability. The effect of MID1 E3 ligase activity within the cell and the role in the pathogenesis of the disease is still not completely unraveled. Here, we report BRAF35, a non-canonical HMG nuclear factor, as a novel MID1 substrate. MID1 is implicated in BRAF35 ubiquitination promoting atypical poly-ubiquitination via K6-, K27- and K29-linkages. We observed a partial co-localization of the two proteins within cytoplasmic bodies. We found that MID1 depletion alters BRAF35 localization in these structures and increases BRAF35 stability affecting its cytoplasmic abundance. Our data reveal a novel role for MID1 and for atypical ubiquitination in balancing BRAF35 presence, and likely its activity, within nuclear and cytoplasmic compartments.

Expanding the SPECC1L mutation phenotypic spectrum to include Teebi hypertelorism syndrome.

Teebi hypertelorism syndrome is a rare autosomal dominant disorder that has eluded a molecular etiology since first described in 1987. Here we report on two unrelated families with a Teebi hypertelorism-like syndrome and Teebi hypertelorism phenotype who have missense mutations in Sperm Antigen With Calponin Homology And Coiled-Coil Domains (SPECC1L), previously associated with oblique facial clefting and Opitz G/BBB syndrome. The first patient and his affected mother were previously-reported by Hoffman et al. in this journal as a new syndrome resembling Teebi hypertelorism and Aarskog syndromes in 2007. This patient had hypertelorism, sagittal and coronal craniosynostosis, ptosis, natal teeth, unusual umbilicus, shawl scrotum, small hands, and feet, with grossly normal development. Our second patient had classic Teebi hypertelorism syndrome with hypertelorism and a giant umbilical hernia. Patient one and his affected mother had a c.1260G>C:p.E420D variant and patient two had a de novo c.1198_1203delATACAC:p.I400_H401del variant in SPECC1L. We review the phenotypic findings in the previously-published Teebi hypertelorism syndrome patients, and the Opitz G/BBB patients with SPECC1L mutations. In addition we emphasize the findings of aortic root dilation and craniosynostosis in these patients, which should be considered in their management.

Surgical management of penoscrotal hypospadias in a child with Opitz G/BBB syndrome: a case report.

Opitz G/BBB syndrome is a rare condition characterized by three significant anomalies; hypertelorism, cleft lip and palate, and hypospadias. However, other anomalies may be associated. Herein, we report a 4-year-old child presented with penoscrotal hypospadias. On examination, hypertelorism and cleft lip and palate were noticed, suggesting a diagnosis of Opitz G/BBB syndrome. The cleft lip was corrected in the first year, and a two-staged surgical approach was implemented for penoscrotal hypospadias. In the first stage, the chordee was corrected and urethral plate was reconstructed using a tabularized incised plate urethroplasty and testicular tunica vaginalis flap. In the second stage, the remanent hypospadias was corrected, and the meatal opening reached its normal location. In conclusion, a two-staged surgical approach for the treatment of penoscrotal hypospadias associated with Opitz G/BBB syndrome may provide excellent outcomes in early-recognized cases. The urologist should pay attention to abnormal facial characteristics in patients with hypospadias.

SPECC1L Mutations Are Not Common in Sporadic Cases of Opitz G/BBB Syndrome.

Opitz G/BBB syndrome (OS) is a rare genetic developmental condition characterized by congenital defects along the midline of the body. The main clinical signs are represented by hypertelorism, laryngo-tracheo-esophageal defects and hypospadias. The X-linked form of the disease is associated with mutations in the MID1 gene located in Xp22 whereas mutations in the SPECC1L gene in 22q11 have been linked to few cases of the autosomal dominant form of this disorder, as well as to other genetic syndromes. In this study, we have undertaken a mutation screening of the SPECC1L gene in samples of sporadic OS cases in which mutations in the MID1 gene were excluded. The heterozygous missense variants identified are already reported in variant databases raising the issue of their pathogenetic meaning. Recently, it was reported that some clinical manifestations peculiar to OS signs are not observed in patients carrying mutations in the SPECC1L gene, leading to the proposal of the designation of 'SPECC1L syndrome' to refer to this disorder. Our study confirms that patients with diagnosis of OS, mainly characterized by the presence of hypospadias and laryngo-tracheo-esophageal defects, do not carry pathogenic SPECC1L mutations. In addition, SPECC1L syndrome-associated mutations are clustered in two specific domains of the protein, whereas the missense variants detected in our work lies elsewhere and the impact of these variants in the function of this protein is difficult to ascertain with the current knowledge and will require further investigations. Nonetheless, our study provides further insight into the SPECC1L syndrome classification.

A novel SPECC1L mutation causing Teebi hypertelorism syndrome: Expanding phenotypic and genetic spectrum.

Only eleven SPECC1L mutations have been reported worldwide which were associated with autosomal dominant oblique facial clefts, Opitz G/BBB Syndrome and Teebi hypertelorism syndrome. In this study, we reported the first Chinese patient with Teebi hypertelorism syndrome. Utilizing whole exome sequencing and Sanger sequencing, we identified a de novo missense mutation NM_015330.3: c.1249A > C, p.(Thr417Pro) in SPECC1L gene. With common manifestations in Teebi hypertelorism syndrome such as special facial appearance, umbilical malformations and congenital heart defects, the patient also had unusual symptoms including recurrent infections, febrile seizures and widely opened anterior fontanelle. Furthermore, all the recorded SPECC1L mutations were analyzed by in silico analysis. Coiled-coil domain 2 was the most frequently mutated domain and positions e and g might be more important than other positions. This paper expanded the phenotypic spectrum of Teebi hypertelorism syndrome and elaborated molecular characteristics of SPECC1L mutations.

A Genetics-First Approach Revealed Monogenic Disorders in Patients With ARM and VACTERL Anomalies.

Background: The VATER/VACTERL association (VACTERL) is defined as the non-random occurrence of the following congenital anomalies: Vertebral, Anal, Cardiac, Tracheal-Esophageal, Renal, and Limb anomalies. As no unequivocal candidate gene has been identified yet, patients are diagnosed phenotypically. The aims of this study were to identify patients with monogenic disorders using a genetics-first approach, and to study whether variants in candidate genes are involved in the etiology of VACTERL or the individual features of VACTERL: Anorectal malformation (ARM) or esophageal atresia with or without trachea-esophageal fistula (EA/TEF). Methods: Using molecular inversion probes, a candidate gene panel of 56 genes was sequenced in three patient groups: VACTERL (n = 211), ARM (n = 204), and EA/TEF (n = 95). Loss-of-function (LoF) and additional likely pathogenic missense variants, were prioritized and validated using Sanger sequencing. Validated variants were tested for segregation and patients were clinically re-evaluated. Results: In 7 out of the 510 patients (1.4%), pathogenic or likely pathogenic variants were identified in SALL1, SALL4, and MID1, genes that are associated with Townes-Brocks, Duane-radial-ray, and Opitz-G/BBB syndrome. These syndromes always include ARM or EA/TEF, in combination with at least two other VACTERL features. We did not identify LoF variants in the remaining candidate genes. Conclusions: None of the other candidate genes were identified as novel unequivocal disease genes for VACTERL. However, a genetics-first approach allowed refinement of the clinical diagnosis in seven patients, in whom an alternative molecular-based diagnosis was found with important implications for the counseling of the families.

Dental treatment of a patient with Opitz G/BBB syndrome.

Opitz G/BBB syndrome is a genetic condition characterized by several abnormalities along the midline of the body, such as hypertelorism, craniofacial deformities, and dysphagia. This study reports the clinical features of Optiz syndrome and its importance in the knowledge of patients who are developmentally challenged as a whole, in order to establish adequate dental treatment for a certain clinical case. A 19-year-old patient visited the Paulista University for a dental treatment. The extraoral examination revealed ocular hypertelorism (wide-spaced eyes), oblique eyelids, epicanthus, low-set cart, and intellectual disability. During the intraoral examination, large caries lesions were observed surrounding the braces of the fixed orthodontic appliance and poor oral hygiene. Preventive and restorative treatments were carried out. It was concluded that the knowledge of patients with special needs as a whole is mandatory for an adequate dental treatment. This is a case report that highlights the importance of dentist and interdisciplinary care attendance for all patient systems, the examination and analyses should not be restricted to the oral cavity.

Two Novel Pathogenic MID1 Variants and Genotype-Phenotype Correlation Reanalysis in X-Linked Opitz G/BBB Syndrome.

X-linked Opitz G/BBB syndrome (XLOS) is a multisystemic congenital condition, caused by mutations in the midline-1 gene (MID1), characterized by a large inter- and intrafamilial phenotypic variability and often associated with intellectual disability (ID). We report clinical, genetic, and molecular findings in 4 patients with typical XLOS dysmorphic features belonging to 2 unrelated families. Two novel pathogenic loss-of-function MID1 variants, a maternally inherited c.1656del and a de novo c.1215_1228dup, were identified. Subsequently, we performed a genotype-phenotype analysis using data from 91 male XLOS patients. To test the mutation impact on the phenotype; the type of mutation, the MID1-impaired domain and function were compared with the presence of each of the major clinical features (hypertelorism, clefts of the lip and/or palate, laryngo-tracheo-esophageal abnormalities, hypospadias and ID) and minor clinical features (brain, heart, and anal defects). No statistically significant correlation was found with these features. Further investigations, as well as exhaustive and unequivocal phenotyping, may be required to improve our knowledge of the biological mechanisms underlying this syndrome and to provide more adequate disease management.

Prenatal diagnosis of maternally inherited X-linked Opitz G/BBB syndrome by chromosomal microarray in a fetus with complex congenital heart disease.

BACKGROUND: Prenatal sonographic diagnosis of Optiz G/BBB syndrome is difficult because the common clinical features, such as hypertelorism, hypospadias and abnormalities of midline structures, including laryngotracheoesophageal defects, are subtle. METHOD: Chromosomal microarray (CMA) analysis using a target enriched Fetal DNA Chip design was performed on the DNA of a fetus with congenital cardiac abnormalities. RESULTS: Fetal DNA chip revealed a 48Kb single copy number loss within chromosome region Xp22.2 (arr[hg18]Xp22.2(10,627,354-10,675,946)x0 mat). This deletion included the 3' UTR region of the MID1 gene predicted to cause the X-linked Opitz G/BBB syndrome. CONCLUSIONS: This case supports the use of CMA in prenatal diagnosis of fetuses with congenital heart disease. CMA allows prenatal diagnosis of genomic aberrations at a much higher resolution compared with conventional karyotyping, and such findings enable proper genetic counseling and decision making in the pregnancy.

A novel mutation in MID1 in a patient with X-linked Opitz G/BBB syndrome.

Opitz G/BBB syndrome (OS) is a genetically heterogeneous disease. We report on an OS patient with a novel inherited mutation in MID1. Metaphase analysis showed a normal male karyotype. Array CGH revealed a maternally inherited duplication at Xp22.31 (6,467,203-7,992,261, hg18), the size was estimated to 1.5Mb. Sequence analysis of the MID1 coding region revealed a novel missense mutation in exon 8 (c.1561C>T/p. R521C) which resulted in an ammonia acid substitution (R521C) in the PRX domain of the MID1 protein. The mutation was inherited from unaffected grandmother and mildly affected mother. Prenatal diagnosis was performed for the third pregnancy after identification of the causative mutation in the family. The third fetus was found to be a female carrier. Postnatal follow-up at 2-month-old showed normal phenotype. In conclusion, we reported a familial OS patient with a novel mutation in exon 8 which provided another evidence for that mutation clustered in C-terminal domain of MID1. The newly identified mutation in our patient expands mutation spectrum in MID1 gene.

Complex rearrangement of the exon 6 genomic region among Opitz G/BBB Syndrome MID1 alterations.

Opitz G/BBB Syndrome (OS) is a multiple congenital anomaly disorder characterized by developmental defects of midline structures. The most relevant clinical signs are ocular hypertelorism, hypospadias, cleft lip and palate, laryngo-tracheo-esophageal abnormalities, imperforate anus, and cardiac defects. Developmental delay, intellectual disability and brain abnormalities are also present. The X-linked form of this disorder is caused by mutations in the MID1 gene coding for a member of the tripartite motif family of E3 ubiquitin ligases. Here, we describe 12 novel patients that carry MID1 mutations emphasizing that laryngo-tracheo-esophageal defects are very common in OS patients and, together with hypertelorism and hypospadias, are the most frequent findings among the full spectrum of OS clinical manifestations. Besides missense and nonsense mutations, small insertions and deletions scattered along the entire length of the gene, we found that a consistent number of MID1 alterations are represented by the deletion of single coding exons. Deep characterization of one of these deletions reveals, for the first time within the MID1 gene, a complex rearrangement composed of two deletions, an inversion and a small insertion that may suggest the involvement of concurrent non-homologous mechanisms in the generation of the observed structural variant.